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1.
Nature ; 573(7774): 421-425, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31511693

RESUMO

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning1. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.


Assuntos
Âmnio/embriologia , Camadas Germinativas/embriologia , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Âmnio/citologia , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Camadas Germinativas/citologia , Humanos , Gravidez , Linha Primitiva/citologia
2.
Annu Rev Physiol ; 83: 359-380, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33035430

RESUMO

The hedgehog (Hh) signaling pathway plays several diverse regulatory and patterning roles during organogenesis of the intestine and in the regulation of adult intestinal homeostasis. In the embryo, fetus, and adult, intestinal Hh signaling is paracrine: Hh ligands are expressed in the endodermally derived epithelium, while signal transduction is confined to the mesenchymal compartment, where at least a dozen distinct cell types are capable of responding to Hh signals. Epithelial Hh ligands not only regulate a variety of mesenchymal cell behaviors, but they also direct these mesenchymal cells to secrete additional soluble factors (e.g., Wnts, Bmps, inflammatory mediators) that feed back to regulate the epithelial cells themselves. Evolutionary conservation of the core Hh signaling pathway, as well as conservation of epithelial/mesenchymal cross talk in the intestine, has meant that work in many diverse model systems has contributed to our current understanding of the role of this pathway in intestinal organogenesis, which is reviewed here.


Assuntos
Proteínas Hedgehog/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Intestinos/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Humanos
3.
Development ; 147(20)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32994164

RESUMO

Between embryonic days 10.5 and 14.5, active proliferation drives rapid elongation of the murine midgut epithelial tube. Within this pseudostratified epithelium, nuclei synthesize DNA near the basal surface and move apically to divide. After mitosis, the majority of daughter cells extend a long, basally oriented filopodial protrusion, building a de novo path along which their nuclei can return to the basal side. WNT5A, which is secreted by surrounding mesenchymal cells, acts as a guidance cue to orchestrate this epithelial pathfinding behavior, but how this signal is received by epithelial cells is unknown. Here, we have investigated two known WNT5A receptors: ROR2 and RYK. We found that epithelial ROR2 is dispensable for midgut elongation. However, loss of Ryk phenocopies the Wnt5a-/- phenotype, perturbing post-mitotic pathfinding and leading to apoptosis. These studies reveal that the ligand-receptor pair WNT5A-RYK acts as a navigation system to instruct filopodial pathfinding, a process that is crucial for continuous cell cycling to fuel rapid midgut elongation.


Assuntos
Sistema Digestório/crescimento & desenvolvimento , Sistema Digestório/metabolismo , Pseudópodes/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Apoptose , Núcleo Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Masculino , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo
4.
Development ; 143(13): 2261-72, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27381224

RESUMO

The vertebrate small intestine requires an enormous surface area to effectively absorb nutrients from food. Morphological adaptations required to establish this extensive surface include generation of an extremely long tube and convolution of the absorptive surface of the tube into villi and microvilli. In this Review, we discuss recent findings regarding the morphogenetic and molecular processes required for intestinal tube elongation and surface convolution, examine shared and unique aspects of these processes in different species, relate these processes to known human maladies that compromise absorptive function and highlight important questions for future research.


Assuntos
Absorção Intestinal , Intestinos/crescimento & desenvolvimento , Animais , Humanos , Microvilosidades/metabolismo , Modelos Biológicos , Morfogênese , Transdução de Sinais
5.
Development ; 143(3): 427-36, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26721501

RESUMO

In the intestine, finger-like villi provide abundant surface area for nutrient absorption. During murine villus development, epithelial Hedgehog (Hh) signals promote aggregation of subepithelial mesenchymal clusters that drive villus emergence. Clusters arise first dorsally and proximally and spread over the entire intestine within 24 h, but the mechanism driving this pattern in the murine intestine is unknown. In chick, the driver of cluster pattern is tensile force from developing smooth muscle, which generates deep longitudinal epithelial folds that locally concentrate the Hh signal, promoting localized expression of cluster genes. By contrast, we show that in mouse, muscle-induced epithelial folding does not occur and artificial deformation of the epithelium does not determine the pattern of clusters or villi. In intestinal explants, modulation of Bmp signaling alters the spatial distribution of clusters and changes the pattern of emerging villi. Increasing Bmp signaling abolishes cluster formation, whereas inhibiting Bmp signaling leads to merged clusters. These dynamic changes in cluster pattern are faithfully simulated by a mathematical model of a Turing field in which an inhibitor of Bmp signaling acts as the Turing activator. In vivo, genetic interruption of Bmp signal reception in either epithelium or mesenchyme reveals that Bmp signaling in Hh-responsive mesenchymal cells controls cluster pattern. Thus, unlike in chick, the murine villus patterning system is independent of muscle-induced epithelial deformation. Rather, a complex cocktail of Bmps and Bmp signal modulators secreted from mesenchymal clusters determines the pattern of villi in a manner that mimics the spread of a self-organizing Turing field.


Assuntos
Padronização Corporal , Proteínas Morfogenéticas Ósseas/metabolismo , Intestinos/embriologia , Microvilosidades/metabolismo , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Epitélio/embriologia , Proteínas Hedgehog/metabolismo , Hibridização In Situ , Ligantes , Mesoderma/embriologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Músculo Liso/embriologia , Tamanho do Órgão , Resistência à Tração
6.
Development ; 143(20): 3711-3722, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27802136

RESUMO

During late gestation, villi extend into the intestinal lumen to dramatically increase the surface area of the intestinal epithelium, preparing the gut for the neonatal diet. Incomplete development of the intestine is the most common gastrointestinal complication in neonates, but the causes are unclear. We provide evidence in mice that Yin Yang 1 (Yy1) is crucial for intestinal villus development. YY1 loss in the developing endoderm had no apparent consequences until late gestation, after which the intestine differentiated poorly and exhibited severely stunted villi. Transcriptome analysis revealed that YY1 is required for mitochondrial gene expression, and ultrastructural analysis confirmed compromised mitochondrial integrity in the mutant intestine. We found increased oxidative phosphorylation gene expression at the onset of villus elongation, suggesting that aerobic respiration might function as a regulator of villus growth. Mitochondrial inhibitors blocked villus growth in a fashion similar to Yy1 loss, thus further linking oxidative phosphorylation with late-gestation intestinal development. Interestingly, we find that necrotizing enterocolitis patients also exhibit decreased expression of oxidative phosphorylation genes. Our study highlights the still unappreciated role of metabolic regulation during organogenesis, and suggests that it might contribute to neonatal gastrointestinal disorders.


Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Organogênese/fisiologia , Fator de Transcrição YY1/metabolismo , Aerobiose/genética , Aerobiose/fisiologia , Animais , Western Blotting , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Organogênese/genética , Fosforilação Oxidativa , Transcriptoma/genética , Fator de Transcrição YY1/genética
7.
Nat Mater ; 16(4): 419-425, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27941807

RESUMO

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However, human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development, thereby helping advance human embryology and reproductive medicine.


Assuntos
Âmnio/metabolismo , Materiais Biomiméticos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-Tronco , Engenharia Tecidual/métodos , Âmnio/citologia , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Medicina Reprodutiva/métodos
8.
Dev Dyn ; 245(5): 614-26, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26930384

RESUMO

BACKGROUND: Digestion is facilitated by coordinated contractions of the intestinal muscularis externa, a bilayered smooth muscle structure that is composed of inner circular muscles (ICM) and outer longitudinal muscles (OLM). We performed transcriptome analysis of intestinal mesenchyme tissue at E14.5, when the ICM, but not the OLM, is present, to investigate the transcriptional program of the ICM. RESULTS: We identified 3967 genes enriched in E14.5 intestinal mesenchyme. The gene expression profiles were clustered and annotated to known muscle genes, identifying a muscle-enriched subcluster. Using publically available in situ data, 127 genes were verified as expressed in ICM. Examination of the promoter and regulatory regions for these co-expressed genes revealed enrichment for cJUN transcription factor binding sites, and cJUN protein was enriched in ICM. cJUN ChIP-seq, performed at E14.5, revealed that cJUN regulatory regions contain characteristics of muscle enhancers. Finally, we show that cJun is a target of Hedgehog (Hh), a signaling pathway known to be important in smooth muscle development, and identify a cJun genomic enhancer that is responsive to Hh. CONCLUSIONS: This work provides the first transcriptional catalog for the developing ICM and suggests that cJun regulates gene expression in the ICM downstream of Hh signaling. Developmental Dynamics 245:614-626, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Intestinos/embriologia , Músculo Liso/embriologia , Transcriptoma , Animais , Genes jun/fisiologia , Proteínas Hedgehog , Camundongos
9.
BMC Dev Biol ; 16: 4, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26912062

RESUMO

BACKGROUND: The Hedgehog (Hh) signaling pathway, acting through three homologous transcription factors (GLI1, GLI2, GLI3) in vertebrates, plays multiple roles in embryonic organ development and adult tissue homeostasis. At the level of the genome, GLI factors bind to specific motifs in enhancers, some of which are hundreds of kilobases removed from the gene promoter. These enhancers integrate the Hh signal in a context-specific manner to control the spatiotemporal pattern of target gene expression. Importantly, a number of genes that encode Hh pathway molecules are themselves targets of Hh signaling, allowing pathway regulation by an intricate balance of feed-back activation and inhibition. However, surprisingly few of the critical enhancer elements that control these pathway target genes have been identified despite the fact that such elements are central determinants of Hh signaling activity. Recently, ChIP studies have been carried out in multiple tissue contexts using mouse models carrying FLAG-tagged GLI proteins (GLI(FLAG)). Using these datasets, we tested whether a meta-analysis of GLI binding sites, coupled with a machine learning approach, could reveal genomic features that could be used to empirically identify Hh-regulated enhancers linked to loci of the Hh signaling pathway. RESULTS: A meta-analysis of four existing GLI(FLAG) datasets revealed a library of GLI binding motifs that was substantially more restricted than the potential sites predicted by previous in vitro binding studies. A machine learning method (kmer-SVM) was then applied to these datasets and enriched k-mers were identified that, when applied to the mouse genome, predicted as many as 37,000 potential Hh enhancers. For functional analysis, we selected nine regions which were annotated to putative Hh pathway molecules and found that seven exhibited GLI-dependent activity, indicating that they are directly regulated by Hh signaling (78% success rate). CONCLUSIONS: The results suggest that Hh enhancer regions share common sequence features. The kmer-SVM machine learning approach identifies those features and can successfully predict functional Hh regulatory regions in genomic DNA surrounding Hh pathway molecules and likely, other Hh targets. Additionally, the library of enriched GLI binding motifs that we have identified may allow improved identification of functional GLI binding sites.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos/genética , Proteínas Hedgehog/genética , Transdução de Sinais/genética , Animais , Sequência de Bases , Linhagem Celular , Proteínas Hedgehog/metabolismo , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Máquina de Vetores de Suporte , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco
10.
Gastroenterology ; 146(1): 157-165.e10, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24120474

RESUMO

BACKGROUND & AIMS: Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. METHODS: We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. RESULTS: Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development. CONCLUSIONS: Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.


Assuntos
Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Liso/embriologia , Miócitos de Músculo Liso/metabolismo , Piloro/embriologia , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição/metabolismo , Animais , Imunofluorescência , Proteína Homeobox Nkx-2.5 , Camundongos , Músculo Liso/metabolismo , Piloro/metabolismo
11.
Development ; 139(3): 488-97, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22190634

RESUMO

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.


Assuntos
Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Intestino Delgado/citologia , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Caliciformes/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Celulas de Paneth/metabolismo , Regiões Promotoras Genéticas , Receptor Notch1/antagonistas & inibidores , Receptor Notch2/antagonistas & inibidores , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/fisiologia
12.
Proc Natl Acad Sci U S A ; 109(39): 15817-22, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-23019366

RESUMO

In the adult intestine, an organized array of finger-like projections, called villi, provide an enormous epithelial surface area for absorptive function. Villi first emerge at embryonic day (E) 14.5 from a previously flat luminal surface. Here, we analyze the cell biology of villus formation and examine the role of paracrine epithelial Hedgehog (Hh) signals in this process. We find that, before villus emergence, tight clusters of Hh-responsive mesenchymal cells form just beneath the epithelium. Cluster formation is dynamic; clusters first form dorsally and anteriorly and spread circumferentially and posteriorly. Statistical analysis of cluster distribution reveals a patterned array; with time, new clusters form in spaces between existing clusters, promoting approximately four rounds of villus emergence by E18.5. Cells within mesenchymal clusters express Patched1 and Gli1, as well as Pdgfrα, a receptor previously shown to participate in villus development. BrdU-labeling experiments show that clusters form by migration and aggregation of Hh-responsive cells. Inhibition of Hh signaling prevents cluster formation and villus development, but does not prevent emergence of villi in areas where clusters have already formed. Conversely, increasing Hh signaling increases the size of villus clusters and results in exceptionally wide villi. We conclude that Hh signals dictate the initial aspects of the formation of each villus by controlling mesenchymal cluster aggregation and regulating cluster size.


Assuntos
Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Hedgehog/genética , Humanos , Mucosa Intestinal/citologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Transgênicos , Receptores Patched , Receptor Patched-1 , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteína GLI1 em Dedos de Zinco
13.
Dev Biol ; 382(1): 82-97, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23916850

RESUMO

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.


Assuntos
Epitélio/embriologia , Epitélio/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Papilas Gustativas/embriologia , Papilas Gustativas/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Compartimento Celular , Linhagem da Célula , Proliferação de Células , Microambiente Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fatores de Transcrição Kruppel-Like/metabolismo , Ligantes , Mesoderma/citologia , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/metabolismo , Papilas Gustativas/citologia , Papilas Gustativas/ultraestrutura , Fatores de Tempo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco
14.
Am J Physiol Gastrointest Liver Physiol ; 307(4): G430-6, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24970776

RESUMO

The gastric ligaments are superficial cord-like structures, located on the lesser curvature of the stomach, that extend from the pylorus to the esophagus. These ligaments have been documented in a wide variety of mammalian species, including humans, but their composition and ontogeny is unexplored. Here, we demonstrate that, during ontogeny, the gastric ligaments are first visible as extensions from a C-shaped domain of Gata3-expressing cells that surround the future pylorus; this domain will later give rise to the pyloric outer longitudinal muscle (OLM). The open ends of the C are located ventrally, and, beginning at embryonic day (E) 13.5, the ligaments grow anteriorly from these points. Whereas most other ligaments of the stomach are neurovascular in nature, the gastric ligaments are composed of smooth muscle cells that mature between E14.5 and E16.5. The gastric ligaments coexpress the transcription factors Gata3, Nkx2-5, and Sox9, and germline loss of Gata3 or conditional deletion of Nkx2-5 abrogates Sox9 expression and impairs gastric ligament smooth muscle development; similar phenotypes were previously seen in the OLM. In accord with this molecular contiguity between the OLM and gastric ligaments, three-dimensional image reconstruction highlights physical contiguity between these smooth muscle structures, suggesting that they may work together as a unit to control flexure of the pyloric region, a function similar to the ligament of Treitz at the duodenojejunal junction. These findings may have implications for our understanding of normal pyloric sphincter function, as well as the common human congenital pathology idiopathic hypertrophic pyloric stenosis.


Assuntos
Fator de Transcrição GATA3/fisiologia , Proteínas de Homeodomínio/fisiologia , Ligamentos/embriologia , Músculo Liso/embriologia , Fatores de Transcrição/fisiologia , Animais , Proteína Homeobox Nkx-2.5 , Ligamentos/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição SOX9/biossíntese
15.
Gastroenterology ; 144(7): 1478-87, 1487.e1-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23470617

RESUMO

BACKGROUND & AIMS: Stress alters brain-gut interactions and could exacerbate intestinal disorders, including irritable bowel syndrome. Alterations in the intestinal microbiota have been associated with irritable bowel syndrome. Maintenance of healthy microbiota requires nucleotide-binding oligomerization domain protein-like receptors, pyrin-domain containing (NLRP)-6 inflammasomes. We investigated the involvement of NLRP6 in water-avoidance stress (WAS)-induced intestinal disorders in mice. METHODS: B57BL6 mice were subjected to WAS for 1 hour each day for 10 days; body weights and intestinal inflammation and permeability were analyzed. We investigated signaling via the NLRP3 and NLRP6 inflammasomes, and the role of corticotropin-releasing hormone (CRH) in WAS-associated inflammation and NLRP6 inhibition. Mice that were not exposed to stress were co-housed with mice subjected to WAS to determine the effects of WAS-induced dysbiosis, measured by sequencing bacterial 16S ribosomal RNA. We also assessed the effects of a peroxisome proliferator-activated receptor-γ agonist and probiotics. RESULTS: WAS-induced small-bowel inflammation (enteritis) was associated with inhibition of NLRP6, but not NLRP3, and was prevented by a peroxisome proliferator-activated receptor-γ agonist, which induced epithelial expression of NLRP6. CRH was released during WAS and inhibited NLRP6 expression. WAS induced alterations in the gut microbiota of mice; co-housed nonstressed mice developed enteritis associated with increased CRH and decreased levels of NLRP6. Probiotic therapy reduced intestinal inflammation in mice with WAS-induced enteritis. CONCLUSIONS: Exposure of mice to stress inhibits NLRP6 and alters the composition of the gut microbiota, leading to intestinal inflammation. These findings might explain the benefits of probiotics for patients with stress-associated gastrointestinal disorders.


Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Enterite/etiologia , Receptores de Superfície Celular/fisiologia , Estresse Psicológico/fisiopatologia , Animais , Modelos Animais de Doenças , Enterite/terapia , Feminino , Inflamassomos/metabolismo , Síndrome do Intestino Irritável/fisiopatologia , Síndrome do Intestino Irritável/psicologia , Metagenoma/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/agonistas , Probióticos/uso terapêutico , Estresse Psicológico/complicações
16.
Development ; 138(20): 4423-32, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21880782

RESUMO

The cellular mechanisms that drive growth and remodeling of the early intestinal epithelium are poorly understood. Current dogma suggests that the murine fetal intestinal epithelium is stratified, that villi are formed by an epithelial remodeling process involving the de novo formation of apical surface at secondary lumina, and that radial intercalation of the stratified cells constitutes a major intestinal lengthening mechanism. Here, we investigate cell polarity, cell cycle dynamics and cell shape in the fetal murine intestine between E12.5 and E14.5. We show that, contrary to previous assumptions, this epithelium is pseudostratified. Furthermore, epithelial nuclei exhibit interkinetic nuclear migration, a process wherein nuclei move in concert with the cell cycle, from the basal side (where DNA is synthesized) to the apical surface (where mitosis takes place); such nuclear movements were previously misinterpreted as the radial intercalation of cells. We further demonstrate that growth of epithelial girth between E12.5 and E14.5 is driven by microtubule- and actinomyosin-dependent apicobasal elongation, rather than by progressive epithelial stratification as was previously thought. Finally, we show that the actin-binding protein Shroom3 is crucial for the maintenance of the single-layered pseudostratified epithelium. In mice lacking Shroom3, the epithelium is disorganized and temporarily stratified during villus emergence. These results favor an alternative model of intestinal morphogenesis in which the epithelium remains single layered and apicobasally polarized throughout early intestinal development.


Assuntos
Mucosa Intestinal/embriologia , Animais , Ciclo Celular , Polaridade Celular , Forma Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Morfogênese , Gravidez
17.
Dev Biol ; 361(1): 39-56, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22024319

RESUMO

Although canonical Wnt signaling is known to regulate taste papilla induction and numbers, roles for noncanonical Wnt pathways in tongue and taste papilla development have not been explored. With mutant mice and whole tongue organ cultures we demonstrate that Wnt5a protein and message are within anterior tongue mesenchyme across embryo stages from the initiation of tongue formation, through papilla placode appearance and taste papilla development. The Wnt5a mutant tongue is severely shortened, with an ankyloglossia, and lingual mesenchyme is disorganized. However, fungiform papilla morphology, number and innervation are preserved, as is expression of the papilla marker, Shh. These data demonstrate that the genetic regulation for tongue size and shape can be separated from that directing lingual papilla development. Preserved number of papillae in a shortened tongue results in an increased density of fungiform papillae in the mutant tongues. In tongue organ cultures, exogenous Wnt5a profoundly suppresses papilla formation and simultaneously decreases canonical Wnt signaling as measured by the TOPGAL reporter. These findings suggest that Wnt5a antagonizes canonical Wnt signaling to dictate papilla number and spacing. In all, distinctive roles for Wnt5a in tongue size, fungiform papilla patterning and development are shown and a necessary balance between non-canonical and canonical Wnt paths in regulating tongue growth and fungiform papillae is proposed in a model, through the Ror2 receptor.


Assuntos
Mesoderma/metabolismo , Transdução de Sinais/fisiologia , Papilas Gustativas/embriologia , Língua/embriologia , Proteínas Wnt/metabolismo , Animais , Western Blotting , Bromodesoxiuridina , Feminino , Galactosídeos , Proteínas Hedgehog/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Indóis , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Ratos , Ratos Sprague-Dawley , Papilas Gustativas/metabolismo , Proteína Wnt-5a
18.
Am J Pathol ; 181(6): 2114-25, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23036899

RESUMO

Gastric adenocarcinoma is one of the leading causes of cancer mortality worldwide. It arises through a stepwise process that includes prominent inflammation with expression of interferon-γ (IFN-γ) and multiple other pro-inflammatory cytokines. We engineered mice expressing IFN-γ under the control of the stomach-specific H(+)/K(+) ATPase ß promoter to test the potential role of this cytokine in gastric tumorigenesis. Stomachs of H/K-IFN-γ transgenic mice exhibited inflammation, expansion of myofibroblasts, loss of parietal and chief cells, spasmolytic polypeptide expressing metaplasia, and dysplasia. Proliferation was elevated in undifferentiated and metaplastic epithelial cells in H/K-IFN-γ transgenic mice, and there was increased apoptosis. H/K-IFN-γ mice had elevated levels of mRNA for IFN-γ target genes and the pro-inflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α. Intracellular mediators of IFN-γ and IL-6 signaling, pSTAT1 and pSTAT3, respectively, were detected in multiple cell types within stomach. H/K-IFN-γ mice developed dysplasia as early as 3 months of age, and 4 of 39 mice over 1 year of age developed antral polyps or tumors, including one adenoma and one adenocarcinoma, which expressed high levels of nuclear ß-catenin. Our data identified IFN-γ as a pivotal secreted factor that orchestrates complex changes in inflammatory, epithelial, and mesenchymal cell populations to drive pre-neoplastic progression in stomach; however, additional alterations appear to be required for malignant conversion.


Assuntos
Mucosa Gástrica/metabolismo , Inflamação/patologia , Interferon gama/genética , Estômago/patologia , Animais , Apoptose/genética , Atrofia , Linhagem da Célula/genética , Proliferação de Células , Progressão da Doença , Feminino , ATPase Trocadora de Hidrogênio-Potássio/genética , Proteínas Hedgehog/metabolismo , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intercelular , Interferon gama/metabolismo , Masculino , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamanho do Órgão , Células Parietais Gástricas/metabolismo , Células Parietais Gástricas/patologia , Peptídeos/metabolismo , Lesões Pré-Cancerosas/patologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Regulação para Cima/genética
19.
Dev Biol ; 355(1): 152-62, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21545794

RESUMO

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.


Assuntos
Proteínas Hedgehog/metabolismo , Homeostase , Mucosa Intestinal/metabolismo , Músculo Liso/fisiologia , Proteínas Nucleares/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/fisiologia , Proteínas Hedgehog/genética , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/fisiologia , Mesoderma , Camundongos , Camundongos Transgênicos , Miofibroblastos , Proteína GLI1 em Dedos de Zinco
20.
Gastroenterology ; 138(7): 2368-77, 2377.e1-4, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20206176

RESUMO

BACKGROUND & AIMS: Epithelial Hedgehog (Hh) ligands regulate several aspects of fetal intestinal organogenesis, and emerging data implicate the Hh pathway in inflammatory signaling in the adult colon. Here, we investigated the effects of chronic Hh inhibition in vivo and profiled molecular pathways acutely modulated by Hh signaling in the intestinal mesenchyme. METHODS: The progression of inflammatory disease was characterized in a bi-transgenic mouse model of chronic Hh inhibition (VFHhip). In parallel, microarray and bioinformatic analyses (Gene Ontology terms overrepresentation analysis, hierarchical clustering, and MeSH term filtration) were performed on isolated cultured intestinal mesenchyme acutely exposed to Hh ligand. RESULTS: Six- to 10-month-old VFHhip animals exhibited villus smooth muscle loss and subsequent villus atrophy. Areas of villus loss became complicated by spontaneous inflammation and VFHhip animals succumbed to wasting and death. Phenotypic similarities were noted between the VFHhip phenotype and human inflammatory disorders, especially human celiac disease. Microarray analysis revealed that inflammatory pathways were acutely activated in intestinal mesenchyme cultured in the absence of epithelium, and the addition of Hh ligand alone was sufficient to largely reverse this inflammatory response within 24 hours. CONCLUSIONS: Hh ligand is a previously unrecognized anti-inflammatory epithelial modulator of the mesenchymal inflammatory milieu. Acute modulation of Hh signals results in changes in inflammatory pathways in intestinal mesenchyme, while chronic inhibition of Hh signaling in adult animals leads to spontaneous intestinal inflammation and death. Regulation of epithelial Hh signaling may be an important mechanism to modulate tolerogenic versus proinflammatory signaling in the small intestine.


Assuntos
Enterite/prevenção & controle , Proteínas Hedgehog/fisiologia , Mucosa Intestinal/imunologia , Transdução de Sinais/fisiologia , Animais , Antígeno CD11b/análise , Enterite/etiologia , Regulação da Expressão Gênica , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Células Mieloides/imunologia
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